L-methionine which is one of the essential amino acids in vivo is contained in most of proteins and is present in a free state in soy sauce which is one of seasoning. It is widely used as feed and food additives, and is also used as infusion solution for medical usage or raw material for synthesizing medicines. Methionine is an important amino acid involved in methyl transfer reaction in vivo. First, by reacting with ATP, methionine is converted to δ-adenosylmethionine, which then donates methyl group to various acceptors; and then it is converted to cysteine via homocysteine and cystathionine. A red bread mold synthesizes methionine from cysteine. A scent of fermented foods such as soy sauce or cheese is often due to aldehyde, alcohol, and/or ester derived from methionine.
Also, methionine acts as a precursor of the compounds such as choline, lecithin, and creatine, and is used as raw material for synthesizing cysteine and taurine, and serving as a sulfur donor. In addition, methionine is associated with the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) inhibit accumulation of fat in liver and arteries in vivo, and take various roles such as alleviating depression, inflammation, liver disease, and muscle pain. Furthermore, methionine and/or S-adenosyl-L-methionine provide various functions such as inhibiting fat deposition in a liver, which promotes fat metabolism, and arteries, increasing bloodstream circulation in a brain, heart, and kidney, stimulating digestion, promoting detoxification and excretion of toxic substances, and promoting the excretion of heavy metals such as lead. It has been reported that daily intake of 800 to 1,600 mg of methionine exhibits superior antidepressant effect, improvement of liver function in patients with liver diseases, particularly liver diseases caused by alcohol, excellent anti-inflammatory effect in bone joint disease, and stimulation of joint recovery in the same. Also, as an essential nutrient for hair, methionine is known to provide nourishment to the brittle hair, preventing hair loss.
For a chemical synthesis of methionine, most commonly L-methionine is produced through hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. However, when methionine is produced by such chemical synthesis process, there is disadvantage of producing mixed forms of L-type and D-type. In this regard, there is a patent disclosing the technique that allows a selective production of L-methionine using a biological approach (WO2008/013432). This method, named simply as a two-step method, comprises a process of converting the L-methionine precursor by fermentation and a process of converting the L-methionine precursor to L-methionine by using enzymes. The type of L-methionine precursor preferably comprises O-acetylhomoserine and O-succinylhomoserine. Development of the two-step method resolves all the existing problems such as toxicity of a substrate specific to sulfide, feedback control in a strain by methionine and SAM, and decomposition of intermediate product specific to cystathionine gamma synthase, O-succinylhomoserine sulfhydrylase, and O-acetylhomoserine sulfhydrylase. Furthermore, this method, which produces only L-methionine selectively, is superior to the conventional chemical synthesis process that produces both of D-methionine and L-methionine simultaneously, and this method can additionally produce organic acid, more specifically, succinic acid and acetic acid, as by-product through the same reaction.
The enzymatic conversion process in the two-step method employs the enzymes having cystathionine gamma synthase activity, O-succinylhomoserine sulfhydrylase activity or O-acetylhomoserine sulfhydrylase activity, and produces L-methionine and organic acids through enzyme reaction of O-acetylhomoserine or O-succinylhomoserine, which is a precursor of L-methionine, with methyl mercaptan.
In the enzymatic conversion reaction for producing L-methionine from O-acetylhomoserine, which is a precursor of L-methionine, various microbial-derived O-acetylhomoserine sulfhydrylases may be used. However, in order to be used as industrial convertase, the enzyme needs to meet several requirements to maximize cost-effectiveness. Firstly, the enzyme should have the characteristics of high activity and high conversion rate, and the overexpression thereof should be possible in E. coli. In general, for the reactions that use a purified enzyme, the activity of enzyme, reaction rate, and high affinity to a substrate are essential. But, if enzyme homogenate is added directly into the reaction, the overexpression of enzyme per unit cell should be possible in addition to having a high activity, in order to proceed the reaction with a minimal amount of homogenate. Secondly, the enzyme needs to maintain a high reaction rate with O-acetylhomoserine at high concentration, and the inhibition of its activity should be low even with accumulation of final products L-methionine and acetic acid to high concentration. Lastly, the enzyme should have a thermal stability to maintain its activity during the 1- to 5-hour long reaction. Considering the above requirements, a previously disclosed O-acetylhomoserine sulfhydrylase derived from Hyphomonas neptunium is an excellent enzyme, however in order to maximize the commercial value of the two-step method for producing methionine, a search for the enzymes with enhancement of the three characteristics is necessary.